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Synaptic and metabolic modulation of the bimodal pacemaker activity in the RPal neuron of Helix pomatia L
03OC-9629/79.:lCN-0265SO2.C0/0
Camp. Biochem. Phwiol., Vol. 64A, pp. 265 lo 271 0 Pergamon Press Ltd 1979.Printed in Great Britain
OF SYNAPTIC AND METABOLIC MODULATION THE BIMODAL PACEMAKER ACTIVITY IN THE RPal NEURON OF HELIX POMATIA L. J. SALANKI, KATALIN S.-R~ZSA and I. VADASZ Biological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary (Received 29 January 1979) Abstract-l. The characteristic activity pattern of the bimodal pacemaker neuron (RPal) of Helix porn&a was temporarily inhibited by synaptic input, both in isolated ganglion by electrical stimulation
of nerves and in brain-heart preparation by tactile stimulation of peripheral receptors. 2. Intracellular injection or extracellular application of SH blockers changed bursting of the RPal neuron. Hyperpolarizing phases were absent while the cell generated action potentials in a normal way, or, slow waving was present without generation of action potentials. 3. It is suggested that slow oscillation of the membrane potential is based on an intracellular metabolic cycle, which is independent of spike generation.
lNTRODUCT’lON
The presence of neurons with bimodal pacemaker activity was reported in a number of Gastropoda species, eg. in the CNS of Aplysia (Arvanitaki & Chalazonitis, 1961), Tritoniu (Willows & Hoyle, 1968) Helix (Sakharov & Salanki, 1969), 0th (Gainer, 1972) Heliosoma (Kater & Kaneko, 1972), Onchidium (Katayama, 1973), Achatina (Takeuchi et al., 1975; S-Rozsa, 1979). For the explanation of the bursting activity two different hypotheses were proposed. According to the first (Strumwasser, 1965) a series of action potentials results in Na accumulation inside the cell. This leads to activation of an electrogenic Na-pump which causes hyperpolarization which in turn prevents the generation of spikes. The activity of the sodium pump however results in a decrease of intracellular Na concentration causing inactivation of the pump and elimination of the evoked hyperpolarization. At the critical level of depolarization with generation of action potentials, the cycle starts again. According to this hypothesis the hyperpolarizing phase is comparable to the posttetanic hyperpolarization and bursting is composed of repetition of these cycles. The second hypothesis (Junge & Stephens, 1973; Meech & Standen, 1975) suggests that a slow wave of membrane potential is the consequence of periodic changes in K-permeability of the membrane. According to this, at the generation of action potentials Ca-ions enter the cell, The increased intracellular Ca-concentration causes hyperpolarization and inhibition of spiking by increasing the K-permeability of the membrane. In this phase either by extrusion from the cell or by binding within the cell the intracellular Ca-concentration decreases and as a result both the K-permeability and the resting potential returns to the previous level allowing the spiking again. The two hypothesis agree, that the series of spikes is a prerequisite for the hyperpolarizing phase and further, that the gradual restoration of the previous intracellular ionic concentration results in the decrease of the 265
membrane potential necessary for generation of action potentials. Our previous investigations show that both Na+ and Cazt take part in the spike generation in the RPal neuron of Helix pomatia (Vadasz & Sallnki, 1976), and so either of them can be responsible for the hyperpolarization and for the presence of the slow wave potential in this neuron. Together with this there are a number of data which cannot be explained by either of the above hypotheses, and require some other explanation for the origin of the slow wave. In this paper we are presenting results showing that (1) the generation of action potentials of the bursting neuron does not cause hyperpolarization and, (2) the previous burst of spikes i.e. the entering of Na or Ca ions into the neuron is not a prerequisite for the appearance of the hyperpolarization and slow wave potential. MATERIAL AND METHODS Investigations were carried out on the RPal neuron of Helix pomntia L. identified earlier in the right parietal
ganglion (Sakharov & Salanki, 1969). This neuron gives no branch into the right pallial nerve, originating close to its location from the ganglion, its main axon passing through the visceral ganglion runs into the intestinal nerve. This was proved by simultaneous recording of corresponding action potentials from the soma and the intestinal nerve (S-Rbzsa & Salanki. 1973). and bv intracellular staining with CoCI, (Elekes’et al.,‘l978). ’ For the experiments, an isolated ganglion or half-isolated preparation was used. Resting and action potentials of the neuron were recorded with microelectrodes (2.5 mol KC]). The temperature of the isolated ganglia was kept constant (22°C) in the bath by a Peltier element (V&o, 1974). In cases where the effects of peripheral inputs were investigated in isolated ganglia, the nerves were stimulated by electric pulses. The brain-heart complex was used as halfisolated preparation including the ganglionic ring, n. intestinalis, heart, percicardium and kidney (S.-Rozsa & Salanki, 1973; S.-Rozsa, 1976). The heart was under perfusion with constant pressure. This preparation was used
.I. SAL~NKI. KATALINS-R6z.s~ and I. VADASZ
266
for the investigation of the effect of heart sttmulation to the RPal neuron. For influencing the metabolic process within the neuron. intracellular injection of CdCl, and HgCI, was used. The concentration of substances in the pipette was 100 mmol. Injection was performed by pressure (Meech, 19721, lasting for several seconds. As a maximum 0.5 nl solution was injected into the cell. The extracellular effect of CdC12 was also tested on the RPal neuron. The final concentratron of CdClz in the per-
fusion reached 10m3 and 10e4 mol. RESULTS
Modulation of the bursting by synaptic influences. After isolation of the ganglion in a number of cases the RPal neuron did not show bursting in its activity but fired continuously like a monomodal pacemaker. By keeping the preparation untouched for 10-30 min, the neuron started to produce the characteristic bursting pattern, but in other cases it could not be observed even after several hours. Such a situation occurred more frequently in half-isolated preparations. In brain-heart preparations we found that the characteristic bursting pattern disappeared sometimes spontaneously (Fig. la) and other times as a result of tactile stimulation of the heart, pericardium or kidney. The modulation of bursting was either a lasting inhibition or the cell started firing continuously. The occurrence of the continuous activity is usually preceded by inhibition and so a double phased effect is produced (Fig. lb). In other instances stimulation of the heart did not evoke inhibition in the neuron but increased the frequency of spike generation (Fig. 2). Depending on the duration of the stimulation, the bursting reappears after some period of time, but it can be eliminated or modulated again by repeating the peripheral stimulation. The effects evoked this way are the result of synaptic inputs
A
which were usually well expressed on the mtraccllular recording in the form of EPSPs or IPSPs. but other times synaptic potentials were not recorded from the soma. The bursting of the RPal neuron could be modified also by short electrical stimulation of nerve trunks. which do not contain any branches of the RPal cell. Single pulses applied to the left pallial nerve evoked hyperpolarization in the neuron. This hyperpolarization could interrupt the burst even after the first spike (Fig. 3). This effect could be ascribed clearly to qynaptic input although well expressed synaptic potentials could be observed very rarely. Modification
I.$ the hurstirq
h!, SH
hlockrrs
Intracellular injection of HgClz and CdCl, into the neuron by micropipettes caused various types of changes in bursting. In 20-25 seconds after introduction of 0.1-0.2 nl HgC12 hyperpolarizing phases decreased gradually and finally interburst hyperpolarization ceased totally. The bimodal character of the activity disappeared, only the omission of a spike with more or less regular intervals resembled the previous character of the neuron (Fig. 4). Also the amplitude of the action potentials decreased to about 20”,, within 34min. The injection of 0.1-0.2 nl CdCl, caused somewhat different change in the activity of the neuron. Following injection the hyperpolarizing phases disappeared and a continuous activity lasting for 3WOsec was produced (Fig. Sa). Thereafter the hyperpolarizing phases reappeared with an even greater amplitude with the positive direction in the way that the generation of action potentials started and ended at a lower value of the membrane potential than in the control (Fig. Sb and c). Thus the waving of the membrane potential became more pronounced. In 5 min after injection the amplitude of the spikes became reduced by 50?;,. the frequency of the waving increased and durmg depolarizing phases instead of
b
Fig. I. Bursting activity of RPal neuron of tion occurring “spontaneously”. IPSP-s are time. B-Effect of tactile stimulation of the of the bursting. a--heartbeat, b-the record
He/ivand its change
in brain-heart preparation. A Inhibrwell visible. The bimodal activity was restored in a short heart. Biphasic response, which was followed by restoration of cell activity. Arrow marks the start of heart stimulation.
Modulation of the bimodal pacemaker
267
_-J
50 mV
20 set
Fig. 2. Repeated stimulation of the heart (arrows) did not cause inhibition, but evoked a transient increase in the neuronal activity. a-heartbeat, b-intracellular recording from the neuron.
a series of spikes only doubled spikes were generated (Fig. 5d). Upon hyperpolarizing the neuron artificially the amplitude and frequency of spikes increased while the hyperpolarizing phases became less frequent (Fig. 5e). Upon depolarization the cell produced a continuous activity with low amplitude (Fig. 5f). When higher doses (0.5 nl) of CdClz or HgCI, were injected a rapid reduction of the amplitude of action potentials and decrease of the membrane potential were observed in 5510sec. The neuron became silent and did not respond to stimulation. Extracellular application of CdClz also influenced the bursting neuron but differently than the intracellular one. At perfusing the preparation with lOA mol, there was no change in the hyperpolarizing phases initially, however, the amplitude of the action potentials was reduced very rapidly. The threshold level of spikes appeared to become more depolarized during the burst as compared to the control, and in 20-25 set after Cd application l-2 set long block of repolarization appeared at the beginning of the burst. In about I min the amplitude of the action potentials was reduced by 80 per cent and hyperpolarizing phases were no longer produced (Fig. 6). low3 mol CdCl, caused at the beginning also a decrease in the amplitude of the spikes, and lowering of the membrane potential. Both hyperpolarizing phases and the duration of the burst of spikes became longer, but the last spike of the burst was not followed by immediate repolarization and the cell remained in a depolarized state for about 8-10 sets (Fig. 7). Then fast hyperpolarization took place, creating the conditions favourable for the generation of a new burst of spikes. Such a pattern was present for a long time (lG-20 min), however the number of spikes within the burst and also the amplitude of the potential changes was gradually decreasing. In 354Omin after CdC12 application only a peculiar variation of the membrane potential was present: 2-3 set long periods of depolarization and repolarization with about 10mV amplitudes were alternating, the transit between them is very sharp, but no action potential was present (Fig. 7). DISCUSSION
It has been shown that the bursting activity of the bimodal pacemaker neuron cannot be eliminated by
depolarization or hyperpolarization, because the cell in spite of the current injection in a short time releases from the polarization (Arvanitaki & Chalazonitis, 1968; Gola, 1976). Mlowever, by changing the temperature (Salknki et al., 1973; Wachtel & Wilson, 1973) or the ionic environment (VaddBsz& Salanki, 1976) the neuron looses its characteristic pattern of activity. The present investigations show that the bursting activity of the RPal cell can be modified by other than direct membrane effects. Activation of synaptic inputs changed the bursting very effectively both in brain-heart preparations and in isolated ganglia. This proves that the activity of the Br-type neuron is not independent of peripheral influences, and can be assumed that the bimodal activity dominates only in the absence of inputs which are switched out at isolation of the ganglion. In isolated brain, synaptic potentials are recorded very rarely from this cell. This fact suggests that RPal neuron receives mainly direct inputs from the periphery. Our experiments proved that the peripheral input can be very effective on the cell, because a single stimulus applied to the left pallial nerve was able to stop the burst of spikes which has been started just before the stimulation. Similar results were obtained by Parnas et al. (1974) and Pasic et al. (1976) at shocking different nerves originating from the suboesophageal ganglion, while Johnston & Ayala (1975) and Kreisman er al. (1978)
I2OmV
t
I
t
t
Fig. 3. Effect of stimulation of the left pallial nerve with single electrical pulses (5 V, 10 msec) to the activity of the RPal neuron. Arrows mark stimulation. The stimulus applied during the interburst period (1st and 4th) causes the prolongatjon of the hy~rpol~izing phase, while stimuli applied during a burst (2nd and 3rd) interrupted the generation of action potentials.
J. SAL~NKI, KATALIN S.-Rtizs~ alid 1. VAIGSL
26X
a
b
d
J
2UmV
5 set Fig. 4. Effect of HgCl, injected intracellularly recording. The elimination
(arrow) to the activity of the RPal neuron. of interburst intervals is remarkable.
Continuous
d
Fig. 5. Effect of intraceJhAar Cd& injection (arrow) to the activity of the RPal neuron. b and c are continuous recordings but with different time scale. Between c and d is a 5 set interruption. e--the neuron was hyper~lari~ed by 20mV. f-the neuron was depolarized by 2OmV. e and f were recorded 8-10 min after CdClz injection. Horizontal bar: 10sec for a. b. e. f: 4sec for c. d.
Modulation of the bimodal pacemaker
Fig. 6. Effect of CdC& added into the bath (10m4 mol) to the activity recording.
Arrow
marks
achieved the inhibition of the bursting activity by pharmacons acting at synaptic contacts. The inhibition and consequent irregularities in the cell activity caused by heart stimulation proved that not only the output of the RPal neuron runs through the intestinal nerve, but also inhibitory and excitatory afferents are
Fig. 7. Effect of 10m3 mol CdC12 (added at arrow) are continuous recordings. Interruption
cadmium
of the RPal neuron. application.
269
Continuous
arriving in this nerve to the cell either directly or through interneurons. The long inhibition of activity found in the RPal neuron as a result of series of IPSP-s is a genera1 rule valid for all neurons, however, the long lasting, continuous firing does not agree with the idea that in this particular neuron, the hyper-
to the activity of the RPal neuron. between traces 3 and 4 is 35 min.
Traces
l-3
170
.I.
SAI.ANKI.
KAIALIN S.-R~)ZSA and I. V,\t&/
polarization is evoked by bursts of spikes. The continuous spiking which appears either spontaneously or after peripheral stimulation would not be present if hyperpolarization which normally follows a burst were simply caused by an accumulation of ions during generation of action potentials. Therefore tt seems reasonable to seek an expl~~n~~ti~~ll for the slow waving of the membrane potential in the himodal paccmaker neuron in which the hyperolariration is regarded not as the result of the burst of spikes. At the same time the reappearance of spikes may not be simply the consequence of the elimination of conditions which interpose hyperpolarization between bursts. We suppose that in bursting neurons, some intracellular metabolic cycle is present. which independently of the action potentials. supports the slow waving of the membrane potential. This could be achieved by changing the permeability of the membrane through the intracellLliar activity of ions. This intracellular cycle can run t~mpor~leily or can be influenced by metabolic, synaptic or physical factors (e.g. temperature). The significance of metabolic processes in the generation of bursting was emphasized also by Ayrapetyan (1976). This is supported by our experiments, when both intra- and extracellular application of the SW-blockers, Hg and Cd. inhibited the hyperpolarizing phases partly or totally, while spike generation. although with lowered amplitude. was present for a long time. These experiments prove also that the hyperpolarizing phase is not the consequence of action potentials. Probably SW-blockers. especially when they were injected intracellularly. inhibited the slow waving of the membrane changing its permeability not directly, but through i~~tr~~~~llul~~r processes. Alterations occurring after extraoeliular application of CdCI, in the repolarizing phase of spike generation show that SH-blockers are effective also on the membrane. The block of repolarization occurred either in the first part or at the end of the burst. hut hyperpolarization terminating the burst was very fast and of high degree. This interburst hyperpolarization was preceded in each case by an introductory, smnli. positive potential. This latter component seems to belong to the hyperpolarizing wave, because it was present even in the case when the burst was represented only by a single abortive spike without repolarization (Fig. 7d). It seems that after ;I severe alteration of membrane permeability some component of the slow waving remams. One can suppose that this residual membrane oscillation originates from a less inhibited intracellular cycle, which is responsible normally independently of the spike generation for the slow waving. A similar oscillation with increased frequency was observed also after intracellular CdCl, injection (Fig. Sd), however, in that case the fast membrane processes responsible for spike generation were inhibited to a lower degree. Our results support the idea that membrane oscillation determining the bimodal pacemaker character of the RPal neuron is independent of the spike generation itself. and the initiation both of the depolarizing and of the hyperpolarizing phase is the function of an intracellular metabolic (energetic or enzymatic) cycle. Since both RPal neuron and other Br type neurons are neurosecretory cells. it would be interest-
ing to investigate, whether the productton or I.&XX of neurosecretory substance is connected h> ~nnt’ way with the bimodal pacemaker behavtour 01 thc~~ neurons.
REFEREiiCES ARVAWIAKI A. & CHALAZOK~TISN. (l9hl)
Slow waves and associated spiking in nerve cells of .4p/t%cz. &(I 1!1\t ocdunogr. Monaco 58, 1 15. ARVANITAKI A. & CHALAZONITIS Pi. (19681 Electrical properties and temporal organization in oscillators
and Plenum Press. New York. AYRAPETYANS. N. (1976)Involvement of the sodium pump in slow oscillations underlying the bursting patterns in Helix neurons. In &‘e~rrohio/oq~ of‘ Imvrt~hrcrtcs. GCISWW pnda Brain (Edited by SALANKI J ). pp. 353 370. Akademiai Kiado. Budapest. ELEKES K.. VADASZ I. and SALANKI J. (197X) Ph~si~~lo8ic~il and morphological analysis of the connections of Brtype neurone. .&fu phnioi. Hurig. 51, 102. GAINER H. (1972) Electrouhvsioloeical behavior of an endogenously active nemosecretoiv ceil. Brcriu Ko. 39. 403-4 18. GOI A M. (1976) Electrical properties of bursting pacemaker netnones. in N~t~rohioir)~y! (3’ I~~rw&rtrft~~. &ofropdn Brdn (Edited by SAI.~~SKI J.). pp. 3X1 42.7. Akademiai Kiado. Budapest, JOHNSTON D. & AVALA G. F. (1975) Diphenylhydantoin: action of a common anticonvulsant on bursting pacemaker cell in .4pl~sirr. Srir~~ce 189, 1009- 101 1. JIIN~X D. & STEPHENS C L. (1973) Cyclic variation oi potassium conductance in burst-8ene~ting ncuronr in :lp~r.siii. J. t’fi_r.sictl. (Lo&. t 235, Ii;‘; 181. K~Z.TAI.AMAY. (1973) E~ectrophysiol~~&ic~~l indentificatton 01 neurones and neuml networks in the perioesophapenl ganglion complex of the marine pulmonate mollusc. O~rchiditm~~wrricu/atwn. J. cup. Bioi. 59. 739 75 I
KATER S. B. & KANEIO C. R. S. (1972) An endogenously bursting neuron in the Gastronod mollusc. Ilc/i~itr c~n/(fimiw. ( ‘cmp Ri(rdxm Physiol. 6OC, 145 154. MEEC‘H R. W. (1972) Intracellular calcium injection causes increased potassium conductance in AP(J& nerve cells. Camp. Bioc~~~m.Ph vsiol. 42A. 393-499. PARK& I.. ARMSTRONG D. & STR~:MWASSERF. (1974~ Prolonged excitatory and inhibitory synaptic modulation of a bursting pacemaker neuron. .I hiruroph t:s‘ioi 37, 594--60X. PASIC M.. ZECEVIC D. 81 RISTANOLI(. D. (1976)Prolonged effects of electrical stimulation of the peripheral nerves on a bursting neuron in the snail ganglion. In Nt~urohir~IO!/? c?f’ Insutrhmfes. Gusrropo&~ Brtriti. (Edited by SALANKI J.), pp. 547.-560. Akademiai Kiado. Budapest. S.-R~ZSA K. (1976) Neuronal network underlying the regulation of heart beat in Helix porn&u L. In Naurohioiogl of‘ Itwurtohrutrs. Gusfropodu Bruin. (Edited by SAI ~~NRI J.). pp. 597-613. Akademiai Kiadd. Budapest. S.-Rozsn K. (1979) Heart regulatory neural network in the central nervous system of ~e~ufi~~.~i~~~cff Ferussac (Gastropoda P~Ilmonata). Cmnp. Biockm. Ph~~iol. 63.4, 435445. S.-ROZSA K. & SAL.&NFUJ. (1973) Single neuronc responses to tactile
stimulation
of the heart
in the snail.
pomatiu L. J. camp. Physiol. 84, X7--279.
NC+;\
Modulation of the bimodal pacemaker D. A. & SALANKIJ. (1969) Physiological and pharmacological identification of neurons in the central nervous system of He/ix pomatia L. Acta physiol. hung.
SAKHAROV
35, 19-30.
SAL~NKI J., VA&Z I. & VBR~ M. (1973) Temperature dependence of the activity pattern in the Br-type cell of the snail W&x ~omatia L. Acta ~~~~~0~.kung. 43, 115-124. STRUMWASSER F. (1965) The demonstration and manipulation of circadian rhythm in a single neuron. In Circa&an CIocks(Edited by ASCHOFFJ.), pp* 442-462. North Holland, Amsterdam. TAKEUCHIH., YOKOII., Mom A. & KOHSAKAM. (1975) Effects on nucleic acid components and their relatives on the excitability of dopamine sensitive giant neurones, identified in suboesophageal ganglia of the African giant snail (Achatina fulica Ferussac). Gen. pharmac. 6, 77-85.
VADASZ I.& SAL~NKIJ. (1976) Mechanisms
271
of spike and burst generation in the bimodal pacemaker RPal neuron of Helix pomatia L. In Neurobiolog ofInvertebrates.Gastropoda Brain. (Edited by SAL~~~KIJ.), pp. 371-380. Akadtmiai Kiado,? Budapest. Vt~6 M. (1974) Temperature control network for the investigation of nerve cells. Annts, Inst. biol. Tihany, 41, t 19-126. WACHTELH. & WILXINW. H. (1973) Voltage clamp analysis of rhythmic slow wave generation in bursting neurons. In Neurobiology of Invertebrates. Mechanisms of rhythm regulation (Edited by SALANKI J.), pp. 59-80. Akademiai Kiado, Budapest. WXLLOWS A. 0. D. & HOYLEG. (1968) Correlation of behaviour with the activity of single identifiable neurons in the brain of Tritonia. In Neurob~oiogy of l~vertebrates (Edited by SALANKIJ.), pp. 443461. Akadtmiai Kiadb, Budapest and Plenum Press, New York.
Camp. Biochem. Phwiol., Vol. 64A, pp. 265 lo 271 0 Pergamon Press Ltd 1979.Printed in Great Britain
OF SYNAPTIC AND METABOLIC MODULATION THE BIMODAL PACEMAKER ACTIVITY IN THE RPal NEURON OF HELIX POMATIA L. J. SALANKI, KATALIN S.-R~ZSA and I. VADASZ Biological Research Institute of the Hungarian Academy of Sciences, H-8237 Tihany, Hungary (Received 29 January 1979) Abstract-l. The characteristic activity pattern of the bimodal pacemaker neuron (RPal) of Helix porn&a was temporarily inhibited by synaptic input, both in isolated ganglion by electrical stimulation
of nerves and in brain-heart preparation by tactile stimulation of peripheral receptors. 2. Intracellular injection or extracellular application of SH blockers changed bursting of the RPal neuron. Hyperpolarizing phases were absent while the cell generated action potentials in a normal way, or, slow waving was present without generation of action potentials. 3. It is suggested that slow oscillation of the membrane potential is based on an intracellular metabolic cycle, which is independent of spike generation.
lNTRODUCT’lON
The presence of neurons with bimodal pacemaker activity was reported in a number of Gastropoda species, eg. in the CNS of Aplysia (Arvanitaki & Chalazonitis, 1961), Tritoniu (Willows & Hoyle, 1968) Helix (Sakharov & Salanki, 1969), 0th (Gainer, 1972) Heliosoma (Kater & Kaneko, 1972), Onchidium (Katayama, 1973), Achatina (Takeuchi et al., 1975; S-Rozsa, 1979). For the explanation of the bursting activity two different hypotheses were proposed. According to the first (Strumwasser, 1965) a series of action potentials results in Na accumulation inside the cell. This leads to activation of an electrogenic Na-pump which causes hyperpolarization which in turn prevents the generation of spikes. The activity of the sodium pump however results in a decrease of intracellular Na concentration causing inactivation of the pump and elimination of the evoked hyperpolarization. At the critical level of depolarization with generation of action potentials, the cycle starts again. According to this hypothesis the hyperpolarizing phase is comparable to the posttetanic hyperpolarization and bursting is composed of repetition of these cycles. The second hypothesis (Junge & Stephens, 1973; Meech & Standen, 1975) suggests that a slow wave of membrane potential is the consequence of periodic changes in K-permeability of the membrane. According to this, at the generation of action potentials Ca-ions enter the cell, The increased intracellular Ca-concentration causes hyperpolarization and inhibition of spiking by increasing the K-permeability of the membrane. In this phase either by extrusion from the cell or by binding within the cell the intracellular Ca-concentration decreases and as a result both the K-permeability and the resting potential returns to the previous level allowing the spiking again. The two hypothesis agree, that the series of spikes is a prerequisite for the hyperpolarizing phase and further, that the gradual restoration of the previous intracellular ionic concentration results in the decrease of the 265
membrane potential necessary for generation of action potentials. Our previous investigations show that both Na+ and Cazt take part in the spike generation in the RPal neuron of Helix pomatia (Vadasz & Sallnki, 1976), and so either of them can be responsible for the hyperpolarization and for the presence of the slow wave potential in this neuron. Together with this there are a number of data which cannot be explained by either of the above hypotheses, and require some other explanation for the origin of the slow wave. In this paper we are presenting results showing that (1) the generation of action potentials of the bursting neuron does not cause hyperpolarization and, (2) the previous burst of spikes i.e. the entering of Na or Ca ions into the neuron is not a prerequisite for the appearance of the hyperpolarization and slow wave potential. MATERIAL AND METHODS Investigations were carried out on the RPal neuron of Helix pomntia L. identified earlier in the right parietal
ganglion (Sakharov & Salanki, 1969). This neuron gives no branch into the right pallial nerve, originating close to its location from the ganglion, its main axon passing through the visceral ganglion runs into the intestinal nerve. This was proved by simultaneous recording of corresponding action potentials from the soma and the intestinal nerve (S-Rbzsa & Salanki. 1973). and bv intracellular staining with CoCI, (Elekes’et al.,‘l978). ’ For the experiments, an isolated ganglion or half-isolated preparation was used. Resting and action potentials of the neuron were recorded with microelectrodes (2.5 mol KC]). The temperature of the isolated ganglia was kept constant (22°C) in the bath by a Peltier element (V&o, 1974). In cases where the effects of peripheral inputs were investigated in isolated ganglia, the nerves were stimulated by electric pulses. The brain-heart complex was used as halfisolated preparation including the ganglionic ring, n. intestinalis, heart, percicardium and kidney (S.-Rozsa & Salanki, 1973; S.-Rozsa, 1976). The heart was under perfusion with constant pressure. This preparation was used
.I. SAL~NKI. KATALINS-R6z.s~ and I. VADASZ
266
for the investigation of the effect of heart sttmulation to the RPal neuron. For influencing the metabolic process within the neuron. intracellular injection of CdCl, and HgCI, was used. The concentration of substances in the pipette was 100 mmol. Injection was performed by pressure (Meech, 19721, lasting for several seconds. As a maximum 0.5 nl solution was injected into the cell. The extracellular effect of CdC12 was also tested on the RPal neuron. The final concentratron of CdClz in the per-
fusion reached 10m3 and 10e4 mol. RESULTS
Modulation of the bursting by synaptic influences. After isolation of the ganglion in a number of cases the RPal neuron did not show bursting in its activity but fired continuously like a monomodal pacemaker. By keeping the preparation untouched for 10-30 min, the neuron started to produce the characteristic bursting pattern, but in other cases it could not be observed even after several hours. Such a situation occurred more frequently in half-isolated preparations. In brain-heart preparations we found that the characteristic bursting pattern disappeared sometimes spontaneously (Fig. la) and other times as a result of tactile stimulation of the heart, pericardium or kidney. The modulation of bursting was either a lasting inhibition or the cell started firing continuously. The occurrence of the continuous activity is usually preceded by inhibition and so a double phased effect is produced (Fig. lb). In other instances stimulation of the heart did not evoke inhibition in the neuron but increased the frequency of spike generation (Fig. 2). Depending on the duration of the stimulation, the bursting reappears after some period of time, but it can be eliminated or modulated again by repeating the peripheral stimulation. The effects evoked this way are the result of synaptic inputs
A
which were usually well expressed on the mtraccllular recording in the form of EPSPs or IPSPs. but other times synaptic potentials were not recorded from the soma. The bursting of the RPal neuron could be modified also by short electrical stimulation of nerve trunks. which do not contain any branches of the RPal cell. Single pulses applied to the left pallial nerve evoked hyperpolarization in the neuron. This hyperpolarization could interrupt the burst even after the first spike (Fig. 3). This effect could be ascribed clearly to qynaptic input although well expressed synaptic potentials could be observed very rarely. Modification
I.$ the hurstirq
h!, SH
hlockrrs
Intracellular injection of HgClz and CdCl, into the neuron by micropipettes caused various types of changes in bursting. In 20-25 seconds after introduction of 0.1-0.2 nl HgC12 hyperpolarizing phases decreased gradually and finally interburst hyperpolarization ceased totally. The bimodal character of the activity disappeared, only the omission of a spike with more or less regular intervals resembled the previous character of the neuron (Fig. 4). Also the amplitude of the action potentials decreased to about 20”,, within 34min. The injection of 0.1-0.2 nl CdCl, caused somewhat different change in the activity of the neuron. Following injection the hyperpolarizing phases disappeared and a continuous activity lasting for 3WOsec was produced (Fig. Sa). Thereafter the hyperpolarizing phases reappeared with an even greater amplitude with the positive direction in the way that the generation of action potentials started and ended at a lower value of the membrane potential than in the control (Fig. Sb and c). Thus the waving of the membrane potential became more pronounced. In 5 min after injection the amplitude of the spikes became reduced by 50?;,. the frequency of the waving increased and durmg depolarizing phases instead of
b
Fig. I. Bursting activity of RPal neuron of tion occurring “spontaneously”. IPSP-s are time. B-Effect of tactile stimulation of the of the bursting. a--heartbeat, b-the record
He/ivand its change
in brain-heart preparation. A Inhibrwell visible. The bimodal activity was restored in a short heart. Biphasic response, which was followed by restoration of cell activity. Arrow marks the start of heart stimulation.
Modulation of the bimodal pacemaker
267
_-J
50 mV
20 set
Fig. 2. Repeated stimulation of the heart (arrows) did not cause inhibition, but evoked a transient increase in the neuronal activity. a-heartbeat, b-intracellular recording from the neuron.
a series of spikes only doubled spikes were generated (Fig. 5d). Upon hyperpolarizing the neuron artificially the amplitude and frequency of spikes increased while the hyperpolarizing phases became less frequent (Fig. 5e). Upon depolarization the cell produced a continuous activity with low amplitude (Fig. 5f). When higher doses (0.5 nl) of CdClz or HgCI, were injected a rapid reduction of the amplitude of action potentials and decrease of the membrane potential were observed in 5510sec. The neuron became silent and did not respond to stimulation. Extracellular application of CdClz also influenced the bursting neuron but differently than the intracellular one. At perfusing the preparation with lOA mol, there was no change in the hyperpolarizing phases initially, however, the amplitude of the action potentials was reduced very rapidly. The threshold level of spikes appeared to become more depolarized during the burst as compared to the control, and in 20-25 set after Cd application l-2 set long block of repolarization appeared at the beginning of the burst. In about I min the amplitude of the action potentials was reduced by 80 per cent and hyperpolarizing phases were no longer produced (Fig. 6). low3 mol CdCl, caused at the beginning also a decrease in the amplitude of the spikes, and lowering of the membrane potential. Both hyperpolarizing phases and the duration of the burst of spikes became longer, but the last spike of the burst was not followed by immediate repolarization and the cell remained in a depolarized state for about 8-10 sets (Fig. 7). Then fast hyperpolarization took place, creating the conditions favourable for the generation of a new burst of spikes. Such a pattern was present for a long time (lG-20 min), however the number of spikes within the burst and also the amplitude of the potential changes was gradually decreasing. In 354Omin after CdC12 application only a peculiar variation of the membrane potential was present: 2-3 set long periods of depolarization and repolarization with about 10mV amplitudes were alternating, the transit between them is very sharp, but no action potential was present (Fig. 7). DISCUSSION
It has been shown that the bursting activity of the bimodal pacemaker neuron cannot be eliminated by
depolarization or hyperpolarization, because the cell in spite of the current injection in a short time releases from the polarization (Arvanitaki & Chalazonitis, 1968; Gola, 1976). Mlowever, by changing the temperature (Salknki et al., 1973; Wachtel & Wilson, 1973) or the ionic environment (VaddBsz& Salanki, 1976) the neuron looses its characteristic pattern of activity. The present investigations show that the bursting activity of the RPal cell can be modified by other than direct membrane effects. Activation of synaptic inputs changed the bursting very effectively both in brain-heart preparations and in isolated ganglia. This proves that the activity of the Br-type neuron is not independent of peripheral influences, and can be assumed that the bimodal activity dominates only in the absence of inputs which are switched out at isolation of the ganglion. In isolated brain, synaptic potentials are recorded very rarely from this cell. This fact suggests that RPal neuron receives mainly direct inputs from the periphery. Our experiments proved that the peripheral input can be very effective on the cell, because a single stimulus applied to the left pallial nerve was able to stop the burst of spikes which has been started just before the stimulation. Similar results were obtained by Parnas et al. (1974) and Pasic et al. (1976) at shocking different nerves originating from the suboesophageal ganglion, while Johnston & Ayala (1975) and Kreisman er al. (1978)
I2OmV
t
I
t
t
Fig. 3. Effect of stimulation of the left pallial nerve with single electrical pulses (5 V, 10 msec) to the activity of the RPal neuron. Arrows mark stimulation. The stimulus applied during the interburst period (1st and 4th) causes the prolongatjon of the hy~rpol~izing phase, while stimuli applied during a burst (2nd and 3rd) interrupted the generation of action potentials.
J. SAL~NKI, KATALIN S.-Rtizs~ alid 1. VAIGSL
26X
a
b
d
J
2UmV
5 set Fig. 4. Effect of HgCl, injected intracellularly recording. The elimination
(arrow) to the activity of the RPal neuron. of interburst intervals is remarkable.
Continuous
d
Fig. 5. Effect of intraceJhAar Cd& injection (arrow) to the activity of the RPal neuron. b and c are continuous recordings but with different time scale. Between c and d is a 5 set interruption. e--the neuron was hyper~lari~ed by 20mV. f-the neuron was depolarized by 2OmV. e and f were recorded 8-10 min after CdClz injection. Horizontal bar: 10sec for a. b. e. f: 4sec for c. d.
Modulation of the bimodal pacemaker
Fig. 6. Effect of CdC& added into the bath (10m4 mol) to the activity recording.
Arrow
marks
achieved the inhibition of the bursting activity by pharmacons acting at synaptic contacts. The inhibition and consequent irregularities in the cell activity caused by heart stimulation proved that not only the output of the RPal neuron runs through the intestinal nerve, but also inhibitory and excitatory afferents are
Fig. 7. Effect of 10m3 mol CdC12 (added at arrow) are continuous recordings. Interruption
cadmium
of the RPal neuron. application.
269
Continuous
arriving in this nerve to the cell either directly or through interneurons. The long inhibition of activity found in the RPal neuron as a result of series of IPSP-s is a genera1 rule valid for all neurons, however, the long lasting, continuous firing does not agree with the idea that in this particular neuron, the hyper-
to the activity of the RPal neuron. between traces 3 and 4 is 35 min.
Traces
l-3
170
.I.
SAI.ANKI.
KAIALIN S.-R~)ZSA and I. V,\t&/
polarization is evoked by bursts of spikes. The continuous spiking which appears either spontaneously or after peripheral stimulation would not be present if hyperpolarization which normally follows a burst were simply caused by an accumulation of ions during generation of action potentials. Therefore tt seems reasonable to seek an expl~~n~~ti~~ll for the slow waving of the membrane potential in the himodal paccmaker neuron in which the hyperolariration is regarded not as the result of the burst of spikes. At the same time the reappearance of spikes may not be simply the consequence of the elimination of conditions which interpose hyperpolarization between bursts. We suppose that in bursting neurons, some intracellular metabolic cycle is present. which independently of the action potentials. supports the slow waving of the membrane potential. This could be achieved by changing the permeability of the membrane through the intracellLliar activity of ions. This intracellular cycle can run t~mpor~leily or can be influenced by metabolic, synaptic or physical factors (e.g. temperature). The significance of metabolic processes in the generation of bursting was emphasized also by Ayrapetyan (1976). This is supported by our experiments, when both intra- and extracellular application of the SW-blockers, Hg and Cd. inhibited the hyperpolarizing phases partly or totally, while spike generation. although with lowered amplitude. was present for a long time. These experiments prove also that the hyperpolarizing phase is not the consequence of action potentials. Probably SW-blockers. especially when they were injected intracellularly. inhibited the slow waving of the membrane changing its permeability not directly, but through i~~tr~~~~llul~~r processes. Alterations occurring after extraoeliular application of CdCI, in the repolarizing phase of spike generation show that SH-blockers are effective also on the membrane. The block of repolarization occurred either in the first part or at the end of the burst. hut hyperpolarization terminating the burst was very fast and of high degree. This interburst hyperpolarization was preceded in each case by an introductory, smnli. positive potential. This latter component seems to belong to the hyperpolarizing wave, because it was present even in the case when the burst was represented only by a single abortive spike without repolarization (Fig. 7d). It seems that after ;I severe alteration of membrane permeability some component of the slow waving remams. One can suppose that this residual membrane oscillation originates from a less inhibited intracellular cycle, which is responsible normally independently of the spike generation for the slow waving. A similar oscillation with increased frequency was observed also after intracellular CdCl, injection (Fig. Sd), however, in that case the fast membrane processes responsible for spike generation were inhibited to a lower degree. Our results support the idea that membrane oscillation determining the bimodal pacemaker character of the RPal neuron is independent of the spike generation itself. and the initiation both of the depolarizing and of the hyperpolarizing phase is the function of an intracellular metabolic (energetic or enzymatic) cycle. Since both RPal neuron and other Br type neurons are neurosecretory cells. it would be interest-
ing to investigate, whether the productton or I.&XX of neurosecretory substance is connected h> ~nnt’ way with the bimodal pacemaker behavtour 01 thc~~ neurons.
REFEREiiCES ARVAWIAKI A. & CHALAZOK~TISN. (l9hl)
Slow waves and associated spiking in nerve cells of .4p/t%cz. &(I 1!1\t ocdunogr. Monaco 58, 1 15. ARVANITAKI A. & CHALAZONITIS Pi. (19681 Electrical properties and temporal organization in oscillators
and Plenum Press. New York. AYRAPETYANS. N. (1976)Involvement of the sodium pump in slow oscillations underlying the bursting patterns in Helix neurons. In &‘e~rrohio/oq~ of‘ Imvrt~hrcrtcs. GCISWW pnda Brain (Edited by SALANKI J ). pp. 353 370. Akademiai Kiado. Budapest. ELEKES K.. VADASZ I. and SALANKI J. (197X) Ph~si~~lo8ic~il and morphological analysis of the connections of Brtype neurone. .&fu phnioi. Hurig. 51, 102. GAINER H. (1972) Electrouhvsioloeical behavior of an endogenously active nemosecretoiv ceil. Brcriu Ko. 39. 403-4 18. GOI A M. (1976) Electrical properties of bursting pacemaker netnones. in N~t~rohioir)~y! (3’ I~~rw&rtrft~~. &ofropdn Brdn (Edited by SAI.~~SKI J.). pp. 3X1 42.7. Akademiai Kiado. Budapest, JOHNSTON D. & AVALA G. F. (1975) Diphenylhydantoin: action of a common anticonvulsant on bursting pacemaker cell in .4pl~sirr. Srir~~ce 189, 1009- 101 1. JIIN~X D. & STEPHENS C L. (1973) Cyclic variation oi potassium conductance in burst-8ene~ting ncuronr in :lp~r.siii. J. t’fi_r.sictl. (Lo&. t 235, Ii;‘; 181. K~Z.TAI.AMAY. (1973) E~ectrophysiol~~&ic~~l indentificatton 01 neurones and neuml networks in the perioesophapenl ganglion complex of the marine pulmonate mollusc. O~rchiditm~~wrricu/atwn. J. cup. Bioi. 59. 739 75 I
KATER S. B. & KANEIO C. R. S. (1972) An endogenously bursting neuron in the Gastronod mollusc. Ilc/i
stimulation
of the heart
in the snail.
pomatiu L. J. camp. Physiol. 84, X7--279.
NC+;\
Modulation of the bimodal pacemaker D. A. & SALANKIJ. (1969) Physiological and pharmacological identification of neurons in the central nervous system of He/ix pomatia L. Acta physiol. hung.
SAKHAROV
35, 19-30.
SAL~NKI J., VA&Z I. & VBR~ M. (1973) Temperature dependence of the activity pattern in the Br-type cell of the snail W&x ~omatia L. Acta ~~~~~0~.kung. 43, 115-124. STRUMWASSER F. (1965) The demonstration and manipulation of circadian rhythm in a single neuron. In Circa&an CIocks(Edited by ASCHOFFJ.), pp* 442-462. North Holland, Amsterdam. TAKEUCHIH., YOKOII., Mom A. & KOHSAKAM. (1975) Effects on nucleic acid components and their relatives on the excitability of dopamine sensitive giant neurones, identified in suboesophageal ganglia of the African giant snail (Achatina fulica Ferussac). Gen. pharmac. 6, 77-85.
VADASZ I.& SAL~NKIJ. (1976) Mechanisms
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of spike and burst generation in the bimodal pacemaker RPal neuron of Helix pomatia L. In Neurobiolog ofInvertebrates.Gastropoda Brain. (Edited by SAL~~~KIJ.), pp. 371-380. Akadtmiai Kiado,? Budapest. Vt~6 M. (1974) Temperature control network for the investigation of nerve cells. Annts, Inst. biol. Tihany, 41, t 19-126. WACHTELH. & WILXINW. H. (1973) Voltage clamp analysis of rhythmic slow wave generation in bursting neurons. In Neurobiology of Invertebrates. Mechanisms of rhythm regulation (Edited by SALANKI J.), pp. 59-80. Akademiai Kiado, Budapest. WXLLOWS A. 0. D. & HOYLEG. (1968) Correlation of behaviour with the activity of single identifiable neurons in the brain of Tritonia. In Neurob~oiogy of l~vertebrates (Edited by SALANKIJ.), pp. 443461. Akadtmiai Kiadb, Budapest and Plenum Press, New York.